A method for determination of glucose 1,6-bisphosphatase

A sensitive and specific method to measure glucose 1,6-bisphosphatase activity, which allows the identification of the reaction products is described. [U-14 C]Glucose 1,6-P2, synthesized by the glucose 1-P kinase activity of phosphofructokinase, is used as substrate. The reaction products are separated and identified by chromatography on ion-exchange paper.     

A method for the determination of glucose synthesis in isolated bovine hepatocytes

A simple method for determining glucose synthesis from radiolabeled precursors in isolated bovine hepatocytes using ion exchange resins is presented. This method allows processing of multiple small volume samples using suspensions of anion and cation exchange resins rather than traditional stacked column separation methods. Hepatocytes were isolated from calf liver by collagenase perfusion of the caudate lobe and were incubated with (14)C-labeled lactate or propionate as gluconeogenic substrates. Glucose synthesis was determined in an aliquot of cell suspension that was vortexed with a slurry of anion exchange (acetate form) resin, followed by a slurry of cation exchange resin. Newly synthesized, labeled glucose was recovered in the supernatant after centrifugation and quantitated by scintillation counting. Using this procedure, more than 98% of the unused labeled precursor was bound to the ion exchange resin and essentially 100% of a labeled glucose tracer was recovered in the supernatant. Pretreatment of hepatocyte suspensions with glucose oxidase was shown to eliminate the accumulation of radioactivity in the supernatant, thus confirming the specificity of this technique for measurement of newly synthesized glucose. This method was sensitive to changes in the rate of hepatic gluconeogenesis that resulted from changes in substrate concentration or the addition of glucagon or fatty acids to the hepatocyte incubations.     

A microcolorimetric assay of inorganic pyrophosphatase

A procedure is described for the assay of inorganic pyrophosphatase in tissues by a microcolorimetric procedure, taking advantage of the marked color intensification of phosphomolybdate by malachite green. Conditions are described for optimum enzyme activity, color stability, and sensitivity. With 1-cm cuvettes the AM660 is 100,000, allowing accurate measurement of Pi in the 1-nmol range. Reaction is conducted at 25 degrees C for 10 min in 0.5 ml of a 50 mM histidine buffer, pH 7.2, containing 0.2 mM inorganic pyrophosphate and 4 mM Mg2+, terminated by addition of 0.05 ml 2.4 M HClO4, cooled in ice, and 0.45 ml of color reagent is added. After standing 10 min at 0 degrees C, the contents are transferred to 1-cm cuvettes and the absorbance is read at 660 nm. Blanks are low, nonenzymatic hydrolysis of PPi is negligible, and color is stable without addition of detergents. The high sensitivity makes this procedure well-adapted to measurement of optimal activities in crude tissue preparations.     

A continuous flow method for the determination of glucose uptake by excised rat diaphragm

A technique is described for the determination of the rate of uptake of glucose by the excised rat diaphragm preparation at intervals as short as 20 seconds. The rate is estimated from the fall in glucose concentration which occurs when a suitable medium flows over the muscle at constant rate. When insulin is added to the perfusion fluid, the rate of glucose uptake rises to a value about 50 per cent higher than that established before the introduction of insulin. Under the conditions described the change in uptake rate requires about 10 to 12 minutes to reach completion, being three-quarters complete in about 7 minutes. Although the major part of this delay must be ascribed to the time required for the washing out of insulin-free medium from the apparatus, and for the diffusion of insulin into the interstitial space, it is suggested that some of the delay may be due to processes occurring between the arrival of insulin at the fibre surface and the exertion of its characteristic effect on glucose uptake, although neither the time-course nor the nature of this induction phase is established.     

A yeast biosensor for glucose determination

Summary A yeast potentiometric biosensor for glucose determination is described. After induction of glycolytic enzyme synthesis a cell suspension of the yeast Hansenula anomala is retained in calcium alginate gel on the surface of a glass electrode. This biosensor gives a Nernstian response in glucose concentration of 5·10^–4–5·10^–3 mol/l with a response time of 5 min and a life-time of at least 2 months. Mannose and fructose are the only significantly interfering substances. The biosensor was used for measurement of glucose concentration in urine with results comparable to those obtained by a photometric enzymatic method.     

A system for the on-line determination of glucose concentration

An off-line glucose analyzer, Yellow Springs Instrument (YSI) model 27 was modified and coupled to various peripheral components to produce a fast, fully automated system for the online determination of glucose concentration. The amount of time required to accomplish each measurement was in the order of two minutes. To demonstrate the utility of this system, various tests were performed. First, a stream containing known amounts of glucose was monitored on-line and the system was calibrated. The calibration curve was shown to be described by Michaelis-Menten kinetics. Once the system was properly calibrated, it was used to monitor the glucose concentration in the effluent stream of two different enzyme reactor systems. The glucose concentrations were within experimental error of those obtained via standard off-line techniques.     

A polarographic method for the simultaneous determination of total glucosinolates and free glucose of cruciferous material

A rapid method for the simultaneous analysis of total free glucose and total glucosinolates in aqueous extracts of cruciferous material is described. The technique, which appears suitable for plant-breeding programs as it allows the processing of more than 100 samples per day, involves the polarographic determination of O2 uptake of free glucose by a system of double-coupled enzymes, such as myrosinase-glucose oxidase. The method has advantages over current methods, because it is very rapid (4 min per analysis), allows two determinations for each analysis, and appears to be very reproducible, accurate, and sensitive.     

葡萄糖测定方法的比较研究

比较了传统斐林定糖,葡萄糖氧化酶－过氧化物酶比色法,葡萄糖氧化酶电极自动分析仪法测定葡萄糖。比较测定了的结果显示,三法的平均标准误差（ＳＤ）,变异系数（ＣＶ）均十分接近。通过对此三种方法的回归相关性分析显示：斐林法－酶终点比色法的回归方程为ｙ＝０．９８４３ｘ＋６．３２３９,相关系数Ｒ＾２＝０．９９８９,斐林法－自动分析仪法的回归方程为ｙ＝１．００８８ｘ＋２．０４８３,相关系数Ｒ＾２＝０．９９９１,